木糖发酵酿酒酵母抑制物耐受能力提升及利用秸秆原料的乙醇发酵性能

Enhancement of inhibitor tolerance during xylose fermentation of Saccharomyces cerevisiae and ethanol fermentation using agricultural straw

木糖利用能力和抑制物耐受能力优良的工业酿酒酵母菌株以及合理的糖化发酵工艺是纤维素燃料乙醇生产的两个关键.对一株工业酿酒酵母菌的磷酸戊糖途径转醛醇酶基因TAL1进行差异过表达,评价其在8种典型抑制物存在时对菌株利用木糖的影响;利用TAL1过表达菌株研究油菜秸秆预处理物料中抑制物含量高低对分步糖化发酵（SHF）、预糖化-同步糖化发酵（P-SSF）和同步糖化发酵（SSF）3种不同糖化发酵方式发酵过程的影响,探讨高固含量发酵的可行性.结果显示,TAL1基因过表达提高了菌株的木糖代谢能力和对8种典型抑制物的耐受能力,适度过表达菌株表现最优,有抑制物存在时的木糖消耗速率提升了20%-70%.秸秆预处理物料中抑制物总含量约为4 g/L时,SHF无法正常发酵,SSF的乙醇收率接近70%,略高于P-SSF;当物料中抑制物总含量下降到约2 g/L时,3种方式都能顺利发酵,SSF表现最优,96 h时的乙醇收率为86.5%,但SSF（96 h）和P-SSF（112 h）所需糖化发酵总时间远低于SHF（144 h）;总固含量约为25%的分批补料-同步糖化发酵（FB-SSF）的乙醇浓度和乙醇收率分别达到54.2 g/L和67.2%.上述结果表明,TAL1基因适度过表达提升了菌株的木糖发酵和抑制物耐受能力,菌株已具备比较优秀的发酵和耐受抑制物的能力;预处理物料中抑制物含量相对较高时采用SSF或P-SSF工艺,而抑制物浓度相对较低时,3种糖化发酵方式都可以采用,但SSF所需发酵时间最短,生产能力最高.

The industrial Saccharomyces cerevisiae strain that has excellent xylose utilization and inhibitor tolerant abilit y as well as rational sacchar if ication and fer mentation process are the t wo key factors in the production oflignocellulosic ethanol. A key gene（transaldolase gene TAL1） involved in the pentose phosphate pathway of the S. cerevisiae strain was overexpressed differentially to evaluate the effect of differential overexpression of TAL1 on the utilization of xylose in the presence of eight typical inhibitors. Using the TAL1-overexpressed strain, the effect of the inhibitor content in pretreated straw on the fermentation efficiencies of three different saccharification and fermentation processes（i.e. separate hydrolysis and fer mentation（SHF）, presaccharif ication-simultaneous saccharif ication and fermentation（P-SSF）, and simultaneous saccharification and fermentation（SSF）） was studied. In addition, the feasibility of high solid load fermentation was investigated. The results showed that the over expression of the TAL1 gene increased the xylose utilizing ability and the tolerance to eight typical inhibitors. The moderate overexpressed strain showed the best performance with an increase of 20%-70% for the xylose consumption rate in the presence of inhibitors. When the total inhibitor content was approximately 4 g/L in the pretreated straw, the ethanol production was difficult during the SHF process, whereas the ethanol yield of SSF was nearly 70%, which was slightly higher than that of P-SSF. When the total inhibitor content in the pretreated straw decreased to approximately 2 g/L, all three processes performed well. SSF had the best performance with an ethanol yield of 86.5% after 96 h fermentation. For SSF（96 h） and P-SSF（112 h）, the total time required for saccharification and fermentation was much shorter than that of SHF（144 h）. In high solid load fermentation（total solid content was 25%） with fed-batch simultaneous saccharification and fermentation, the ethanol concentration and ethanol yield reached 54.2 g/L and 67.2%, respectively. Results revealed that moderate over expression of TAL1 gene effectively improved the xylose utilization and inhibitor tolerance of S. cerevisiae, and the produced strain had excellent abilities of xylose utilization and inhibitor tolerance. When the inhibitor content in the pretreatment straw was high, SSF or P-SSF was preferred, whereas when the inhibitor content in the pretreatment materials was low, any of these three processes was feasible; however, the SSF process had the shortest fermentation time and highest productivity.